Benoît Ho‐Tin‐Noé

The role of platelets in hemostasis is to produce a plug to arrest bleeding. During thrombocytopenia, spontaneous bleeding is seen in some patients but not in others; the reason for this is unknown. Here, we subjected thrombocytopenic mice to models of dermatitis, stroke, and lung inflammation. The mice showed massive hemorrhage that was limited to the area of inflammation and was not observed in uninflamed thrombocytopenic mice. Endotoxin-induced lung inflammation during thrombocytopenia triggered substantial intra-alveolar hemorrhage leading to profound anemia and respiratory distress. By imaging the cutaneous Arthus reaction through a skin window, we observed in real time the loss of vascular integrity and the kinetics of skin hemorrhage in thrombocytopenic mice. Bleeding-observed mostly from venules-occurred as early as 20 minutes after challenge, pointing to a continuous need for platelets to maintain vascular integrity in inflamed microcirculation. Inflammatory hemorrhage was not seen in genetically engineered mice lacking major platelet adhesion receptors or their activators (alphaIIbbeta3, glycoprotein Ibalpha [GPIbalpha], GPVI, and calcium and diacylglycerol-regulated guanine nucleotide exchange factor I [CalDAG-GEFI]), thus indicating that firm platelet adhesion was not necessary for their supporting role. While platelets were previously shown to promote endothelial activation and recruitment of inflammatory cells, they also appear indispensable to maintain vascular integrity in inflamed tissue. Based on our observations, we propose that inflammation may cause life-threatening hemorrhage during thrombocytopenia.

BACKGROUND AND PURPOSE: Neutrophil Extracellular Traps (NETs) are DNA extracellular networks decorated with histones and granular proteins produced by activated neutrophils. NETs have been identified as major triggers and structural factors of thrombosis. A recent study designated extracellular DNA threads from NETs as a potential therapeutic target for improving tissue-type plasminogen activator (tPA)-induced thrombolysis in acute coronary syndrome. The aim of this study was to assess the presence of NETs in thrombi retrieved during endovascular therapy in patients with acute ischemic stroke (AIS) and their impact on tPA-induced thrombolysis. METHODS: We analyzed thrombi from 108 AIS patients treated with endovascular therapy. Thrombi were characterized by hematoxylin/eosin staining, immunostaining, and ex vivo enzymatic assay. Additionally, we assessed ex vivo the impact of deoxyribonuclease 1 (DNAse 1) on thrombolysis of AIS thrombi. RESULTS: Histological analysis revealed that NETs contributed to the composition of all AIS thrombi especially in their outer layers. Quantitative measurement of thrombus NETs content was not associated with clinical outcome or AIS pathogenesis but correlated significantly with endovascular therapy procedure length and device number of passes. Ex vivo, recombinant DNAse 1 accelerated tPA-induced thrombolysis, whereas DNAse 1 alone was ineffective. CONCLUSIONS: This study suggests that thrombus NETs content may be responsible for reperfusion resistance, including mechanical or pharmacological approaches with intravenous tPA, irrespectively of their etiology. The efficacy of a strategy involving an administration of DNAse 1 in addition to tPA should be explored in the setting of AIS. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT02907736.

Cancer is associated with a prothrombogenic state capable of platelet activation. Platelets, on the other hand, can support angiogenesis, a process involved in the progression of tumor growth and metastasis. However, it is unclear whether platelet/tumor interactions substantially contribute to tumor physiology. We investigated whether platelets stabilize tumor vessels and studied the underlying mechanisms. We induced severe acute thrombocytopenia in mice bearing s.c. Lewis lung carcinoma or B16F10 melanoma. Intravital microscopy revealed that platelet depletion led to a rapid destabilization of tumor vessels with intratumor hemorrhage starting as soon as 30 min after induction of thrombocytopenia. Using an inhibitor of glycoprotein Ibalpha (GPIbalpha) and genetically engineered mice with platelet adhesion defects, we investigated the role of platelet adhesion receptors in stabilizing tumor vessels. We found that a single defect in either GPIbalpha, von Willebrand factor, P-selectin, or platelet integrin activation did not lead to intratumor hemorrhage. We then compared the ability of transfused resting and degranulated platelets to prevent intratumor hemorrhage. Whereas resting platelets prevented thrombocytopenia-induced tumor bleeding, circulating degranulated platelets did not. This suggests that the prevention of intratumor hemorrhage by platelets relies on the secretion of the content of platelet granules. Supporting this hypothesis, we further found that thrombocytopenia dramatically impairs the balance between propermeability and antipermeability factors in tumor-bearing animals, in particular depleting blood of angiopoietin-1 and serotonin. Our results show a crucial contribution of platelets to tumor homeostasis through continuous prevention of severe intratumor hemorrhage and consequent cell death. The study also suggests platelet function as a reasonable target for specific destabilization of tumor vessels.

BACKGROUND AND PURPOSE: Downstream microvascular thrombosis (DMT) is known to be a contributing factor to incomplete reperfusion in acute ischemic stroke. The aim of this study was to determine the timing of DMT with intravital imaging and to test the hypothesis that intravenous alteplase infusion could reduce DMT in a transient middle cerebral artery occlusion (MCAO) rat stroke model. METHODS: Rats were subjected to 60-minute transient MCAO. Alteplase (10 mg/kg) was administered 30 minutes after the beginning of MCAO. Real-time intravital fluorescence microscopy through a dura-sparing craniotomy was used to visualize circulating blood cells and fibrinogen. Cerebral microvessel patency was quantitatively evaluated by fluorescein isothiocyanate-dextran perfusion. RESULTS: Immediately after MCAO, platelet and leukocyte accumulation were observed mostly in the venous compartment. Within 30 minutes after MCAO, microthrombi and parietal fibrin deposits were detected in postcapillary microvessels. Alteplase treatment significantly (P=0.006) reduced infarct volume and increased the percentage of perfused vessels during MCAO (P=0.02) compared with saline. Plasma levels of fibrinogen from alteplase-treated rats showed a rapid and profound hypofibrinogenemia. In vitro platelet aggregation demonstrated that alteplase reduced platelet aggregation (P=0.0001) and facilitated platelet disaggregation (P=0.001). These effects were reversible in the presence of exogenous fibrinogen. CONCLUSIONS: Our data demonstrate that DMT is an early phenomenon initiated before recanalization. We further show that alteplase-dependent maintenance of downstream perfusion during MCAO improves acute ischemic stroke outcome through a fibrinogen-dependent platelet aggregation reduction. Our results indicate that early targeting of DMT represents a therapeutic strategy to improve the benefit of large artery recanalization in acute ischemic stroke.